Chikungunya virus (CHIKV) is an alphavirus of the family Togaviridae. It is a positive strand RNA virus that causes a generally non-fatal infection characterized by high fever and sudden onset of polyarthralgia. Hemorrhagic and neurological manifestations including seizures, lymphadenopathy, fulminant hepatitis and conjunctivitis not hitherto associated with CHIKV infections were reported since the re-surgent infection in 2005 (Sourisseau et al., 2007; Kannan et al., 2007). Phylogenetic analyses based on the partial E1 structural glycoprotein sequences have identified three CHIKV lineages, the West African, Asian and the East, Central and South African (ECSA) (Powers et al., 2000). Asian lineage circulated in India and Southeast Asia until it was replaced by the ECSA genotype, which emerged during the 2005-2006 outbreak in the Indian Ocean islands (Yergolkar et al., 2006). Sub-lineages of ECSA strains that had established locally were spread by travellers from endemic areas to Africa, Asia and Europe and caused local outbreaks (Powers and Logue, 2007).
Nearly 1.39 million suspected cases of Chikungunya virus infection occurred in India in 2006. (National Vector Borne Disease Control Programme (NVBDCP), 2007) which was caused by the ECSA strain carrying the E1-226A (Arankalle et al., 2007). The E1-A226V adaptive mutation that increases transmissibility by Aedes albopictus is responsible for the wide geographical spread of the virus since then (de Lamballerie et al., 2008). Host immune pressure and resultant site specific mutations in the human leukocyte antigen (HLA) class-1 restricting elements of CHIKV genome are implicated in the explosive Chikungunya virus outbreaks since 2005 (Tong et al., 2010). Prior art known in the field do not include any vaccine candidate derived from the ECSA strain. Bharat Biotech International Limited has earlier developed (disclosed in WO 2008/026225) the 2006 ECSA strain with E1-226A and its use in the development of potential vaccines against Chikungunya virus infections.
Chikungunya virus strains of the urban (epidemic) transmission cycles show a higher evolutionary rate than that of the enzootic (sylvatic) cycle, and the difference in the evolutionary dynamics between the two transmission cycles are influenced by several factors that determine virus-host interactions such as vector diversity and abundance, vector larval habitats and herd immunity in the population (Volk et al., 2010). Arboviruses like Chikungunya interacts with both the arthropod and the vertebrate hosts, and the selection pressure on the envelope glycoproteins are driven by preferences for vector adaptation and by vertebrate host immune defense mechanisms. Viral evolution tends to select for mutations in the antigenic determinants involved in neutralization as well as those residues involved in vector/host adaptation.
The vaccines under development such as that disclosed in WO 2008030220 and in Akahata et al. 2010 make use of the West African genotype and the E1-A226V isolates. Another CHIKV vaccine development is a DNA vaccine (Mallilankaraman et al., 2011) which is different in scope from that disclosed in this invention. An earlier prototype vaccine which is a live attenuated vaccine used the Asian genotype of the virus (Edelman et al., 2001). DNA vaccines have not been successful in human prophylactic vaccination so far, and live attenuated CHIKV vaccine caused side effects in human subjects (Edelman et al., 2001) who received the vaccine. The CHIKV strain used in the earlier vaccine development (WO 2008/026225) was the 2006 ECSA strain with E1-226A. The strains isolated in 2009-2010 from India as disclosed in this invention belong to a distinct sub-lineage within the ECSA lineage and carry novel mutations in the E2 and E1 envelope glycoproteins. One of the mutations in the E1 glycoprotein in all the isolates reported in the study maps to a region that determines host vector specificity and is under significant positive selection for enhanced adaptation to Adis. aegypti, which is the most abundant mosquito vector in the region and indeed in the tropical countries where prevalence of Chikungunya virus infection is now endemic. Other novel mutations hitherto unreported are also disclosed. Thus it is desirable to make a vaccine composition which would confer immunity to the newly developed and distinct sublineages of the ECSA strain of the Chikungunya virus which would also confer immune protection to the other mutated strains of the ECSA strain propagated by the vector Aedis aegypti. Inventors in this application after prolonged research disclose such an effective vaccine in this application including other additional advantages over the earlier vaccine (WO 2008/026225) such as new methods of inactivation of the virus and improved formulations with novel adjuvants that enhance the immunogenicity of the inactivated viral vaccine and the recombinant subunit vaccines and virosomes which are also included herein this invention.